Foundational Gnotobiotics Concepts


     
Foundational Gnotobiotics Concepts Gnotobiology isn't new, but the dramatic expansion of microbiome research has outpaced the development of a common nomenclature within the gnotobiotic research community. Many different, and sometimes confusing, terms are used when referring to different gnotobiotic concepts. To help, here's an overview of the most commonly used gnotobiotic rodent model concepts with examples of how these concepts are applied in basic research and drug discovery.

Gnotobiology means the study of "known life." Hence, a "gnotobiotic animal" is a host animal harboring only defined (known) microbial life, if any. Gnotobiotic animal models may be either germ-free or be known to carry only a defined set of microorganisms. Somewhat confusingly, "gnotobiotic" is sometimes used in a broader sense to cover mice or rats that were initially germ-free and subsequently colonized, or associated, with a complex microbial community. These communities may be characterized to some extent, but are rarely fully defined, as this would require in-depth analysis of all bacteria, viruses, fungi, protozoa and more.

Conceptual Overview of Gnotobiotic Rodent Models

Concept

Also Known As

Definition

Why Use This Concept

Reductionist approaches
Germ-free Axenic, sterile Germ-free rodents lacking all known organisms (bacteria, fungi, viruses, parasites) except for endogenous retroviruses. The germ-free state is determined within the limitations of the currently available detection methods. Germ-free rodents are considered the gold standard as reliable and reproducible tools for microbiota-association studies and as founders for gnotobiotic or standardized colonies or study cohorts.

Germ-free rodents are additionally crucial as controls in association studies (also called fecal microbiota transplantation).
Mono-associated Monoxenic, mono-colonized Rodents harboring just one organism, e.g. Escherichia coli, Segmented Filamentous Bacteria, or Bacteroides thetaiotaomicron To study the function and effects of a particular organism, e.g. induction of the immune system by the organism1
Di-associated Dixenic, di-colonized, dual-associated, bi-associated, bi-colonized Rodents harboring just two organisms, e.g. B. thetaiotaomicron in conjunction with Faecalibacterium prausnitzii.

The concept may be expanded to tri-associated (trixenic), tetra-associated and so on.
To study synergy, interaction, and adaptation between two organisms and the host2.

To facilitate the colonization with extremely oxygen sensitive bacteria by initial inoculation with an aerotolerant species that prepare an anaerobic enviroment3.
Simplified microbiota-associated Defined flora/microbiota-associated/colonized Rodents harboring a limited number of known (defined) organisms, e.g. the mouse-derived Altered Schaedler Flora or communities with a limited number of human-derived bacteria or from other target species. Such limited consortia typically consist of 5-15 species and may be procured from microbial repositories. For a tractable, time stable, and reproducible model system to study, for example:
  • urinary/plasma metabolic profiles4
  • spatial structure of microbiota5
  • dietary interventions6
  • transkingdom interactions, e.g. between bacteria and phages7
Holistic approaches*
Mouse/rat microbiota-associated with:
- healthy microbiota
- dysbiotic microbiota
- disease model-prone microbiota
- wild-sourced microbiota
Complex microbiota-associated, SPF-colonized, conventionalized Rodents harboring another rodent microbiota than their endogenous microbiota. Typically generated by inoculating germ-free mice or rats, but other approaches such as cross-fostering and embryo transfer may also be used. Phenotype transfer for proof-of-concept hypothesis testing before moving to human patient samples that may more difficult to access8.

To optimize disease model readout and pursue the 3Rs by creating high-responder cohorts9.

To improve translation to humans by creating models with a naturalistic wild mouse microbiota and hence, an improved immune response10.

For establishing rodents with a standardized microbiota to overcome reproducibility issues due to variation in the microbiome over time or between locations.
Human microbiota-associated with:
- healthy microbiota
- dysbiotic microbiota
- patient microbiota
Humanized, complex microbiota-associated, human flora-colonized, microbial xenoengraftment Rodents colonized with human intestinal microbiota, most often from feces.

The proportion of the donor microbiota that is successfully established in the rodents varies and is often smaller than it is for mouse/rat microbiota-associated mice/rats.

The proportion that establish in the animals is stable over time and generations under protected conditions11.

Human microbiota-associated mice should ideally be validated for study of specific immune parameters since immunomodulation of the host is altered in these mice.
For a translational and predictive model to study for example:
  • phenotype transfer to demonstrate causal involvement of microbiome in disease12
  • metabolism of drugs and xenobiotics by the microbiota13
  • pathogen infection14
  • dietary interventions15
  • individualized responses of human microbiotas to develop personalized dietary or drug interventions16
* The holistic approaches are strictly speaking not gnotobiotic unless all members of the complex community are known and precisely defined. While this is rarely the case, these models are nevertheless often referred to as gnotobiotic since they require gnotobiotic or clean IVC housing technology.
Foundational Gnotobiotics Concepts

References:
1. Gaboriau-Routhiau, V.; Rakotobe, S.; Lécuyer, E.; Mulder, I.; Lan, A.; Bridonneau, C.; Rochet, V.; Pisi, A.; De Paepe, M.; Brandi, G.; et al. The key role of segmented filamentous bacteria in the coordinated maturation of gut helper T cell responses. Immunity 2009, 31 (4), 677-689 DOI: 10.1016/j.immuni.2009.08.020.
2. Mahowald, M. A.; Rey, F. E.; Seedorf, H.; Turnbaugh, P. J.; Fulton, R. S.; Wollam, A.; Shah, N.; Wang, C.; Magrini, V.; Wilson, R. K.; et al. Characterizing a model human gut microbiota composed of members of its two dominant bacterial phyla. Proc. Natl. Acad. Sci. 2009, 106 (14), 5859-5864 DOI: 10.1073/pnas.0901529106.
3. Miquel, S.; Leclerc, M.; Martin, R.; Chain, F.; Lenoir, M.; Raguideau, S.; Hudault, S.; Bridonneau, C.; Northen, T.; Bowen, B.; et al. Identification of Metabolic Signatures Linked to Anti-Inflammatory Effects of Faecalibacterium prausnitzii. MBio 2015, 6 (2), e00300-15 DOI: 10.1128/mBio.00300-15.
4. Rezzonico, E.; Mestdagh, R.; Delley, M.; Combremont, S.; Dumas, M.-E.; Holmes, E.; Nicholson, J.; Bibiloni, R. Bacterial adaptation to the gut environment favors successful colonization: microbial and metabonomic characterization of a simplified microbiota mouse model. Gut Microbes 2011, 2 (6), 307-318 DOI: 10.4161/gmic.18754.
5. Mark Welch, J. L.; Hasegawa, Y.; McNulty, N. P.; Gordon, J. I.; Borisy, G. G. Spatial organization of a model 15-member human gut microbiota established in gnotobiotic mice. Proc. Natl. Acad. Sci. 2017, 114 (43), E9105-E9114 DOI: 10.1073/pnas.1711596114.
6. Becker, N.; Kunath, J.; Loh, G.; Blaut, M. Human intestinal microbiota: Characterization of a simplified and stable gnotobiotic rat model. Gut Microbes 2011, 2 (1), 25-33 DOI: 10.4161/gmic.2.1.14651.
7. Reyes, A.; Wu, M.; McNulty, N. P.; Rohwer, F. L.; Gordon, J. I. Gnotobiotic mouse model of phage-bacterial host dynamics in the human gut. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (50), 20236-20241 DOI: 10.1073/pnas.1319470110.
8. Bercik, P.; Denou, E.; Collins, J.; Jackson, W.; Lu, J.; Jury, J.; Deng, Y.; Blennerhassett, P.; Macri, J.; McCoy, K. D.; et al. The intestinal microbiota affect central levels of brain-derived neurotropic factor and behavior in mice. Gastroenterology 2011, 141 (2), 599-609, 609-3 DOI: 10.1053/j.gastro.2011.04.052.
9. Zachariassen, L. F.; Krych, L.; Engkilde, K.; Nielsen, D. S.; Kot, W.; Hansen, C. H. F.; Hansen, A. K. Sensitivity to oxazolone induced dermatitis is transferable with gut microbiota in mice. Submitt. to Sci. Reports 2017.
10. Rosshart, S. P.; Vassallo, B. G.; Angeletti, D.; Hutchinson, D. S.; Morgan, A. P.; Takeda, K.; Hickman, H. D.; McCulloch, J. A.; Badger, J. H.; Ajami, N. J.; et al. Wild Mouse Gut Microbiota Promotes Host Fitness and Improves Disease Resistance. Cell 2017, 0 (0) DOI: 10.1016/j.cell.2017.09.016.
11. Collins, J.; Auchtung, J. M.; Schaefer, L.; Eaton, K. A.; Britton, R. A. Humanized microbiota mice as a model of recurrent Clostridium difficile disease. Microbiome 2015, 3, 35 DOI: 10.1186/s40168-015-0097-2.
12. De Palma, G.; Lynch, M. D. J.; Lu, J.; Dang, V. T.; Deng, Y.; Jury, J.; Umeh, G.; Miranda, P. M.; Pigrau Pastor, M.; Sidani, S.; et al. Transplantation of fecal microbiota from patients with irritable bowel syndrome alters gut function and behavior in recipient mice. Sci. Transl. Med. 2017, 9 (379), eaaf6397 DOI: 10.1126/scitranslmed.aaf6397.
13. Haiser, H. J.; Seim, K. L.; Balskus, E. P.; Turnbaugh, P. J. Mechanistic insight into digoxin inactivation by Eggerthella lenta augments our understanding of its pharmacokinetics. Gut Microbes 2014, 5 (2), 233-238 DOI: 10.4161/gmic.27915.
14. Brooks, P. T.; Brakel, K. A.; Bell, J. A.; Bejcek, C. E.; Gilpin, T.; Brudvig, J. M.; Mansfield, L. S. Transplanted human fecal microbiota enhanced Guillain Barré syndrome autoantibody responses after Campylobacter jejuni infection in C57BL/6 mice. Microbiome 2017, 5 (1), 92 DOI: 10.1186/s40168-017-0284-4.
15. Smits, S. A.; Marcobal, A.; Higginbottom, S.; Sonnenburg, J. L.; Kashyap, P. C. Individualized Responses of Gut Microbiota to Dietary Intervention Modeled in Humanized Mice. mSystems 2016, 1 (5), e00098-16 DOI: 10.1128/mSystems.00098-16.
16. Kuntz, T. M.; Gilbert, J. A.; Venter, J. C.; al., et; Guttmacher, A. E.; al., et; McCarthy, J. J.; al., et; Garraway, L. A.; al., et; et al. Introducing the Microbiome into Precision Medicine. Trends Pharmacol. Sci. 2016, 0 (0), 1304-1351 DOI: 10.1016/j.tips.2016.10.001.